Sucrose/D-Glucose, colorimetric method

Catalogue number: AK00241, 250 tests of each analyte

Application

This rapid and simple specific enzymatic method is used for

the simultaneous determination of sucrose and D-glucose in

foodstuffs, pharmaceuticals, cosmetics and biological

samples.

Introduction

Sucrose and D-glucose occur widely in plant organisms. In

foods, they occur mainly in honey, wine and beer, and a

range of solid foodstuffs such as bread and pastries,

chocolate and candies. In the wine industry, the addition of

sucrose is only allowed in few situations, such as champagne

production.

Principles

The D-glucose concentration is determined before and after

hydrolysis of sucrose by -fructosidase. The sucrose content

is calculated form the difference in D-glucose concentrations

before and after hydrolysis by -fructosidase. Free D-glucose

in the sample extract is determined by conversion to a red

coloured quinoneimine dye compound through the

combined action of glucose oxidase and peroxidase.

Specificity

Using high purity of glucose oxidase, peroxidase and -

fructosidase, this colorimetric method is specific for sucrose

and D-glucose measurement in plant and food extracts. The

colour formed through the reaction is stable at room

temperature for at least 2 hours after development.

Linearity and precision

Linearity of the determination exists from 10 to 100 μg D-

glucose or sucrose per assay (see Figure 1). Standard errors

below 5% are reached routinely.

Kit composition

Solution 1. Buffer (20 mL, pH 4.6). Stable for 2 years at 4 °C.

Dilute the content of bottle 1 to 100 mL with distilled water

befor use. Stable for > 1 year at 4ºC.

Suspension 2. -Fructosidase (5 mL). Stable for 2 years at 4

°C. Swirl bottle before use.

Add 0.02 ml of Suspension 2 plus 0.180 ml of Solution 1, per

assay, to a test tube and homogenise (Solution 1+2). This

solution should be prepared for each assay day.

Solution 3. GOD-POD reagent buffer (2 x 22.5 mL, pH 7.4),

p-hydroxybenzoic acid and sodium azide (0.64% w/v) as a

preservative. Stable for 3 years at 4 °C.

Dilute the contents of one bottle to 0.75 L with distilled water

and use immediately.

Mixture 4. GOD-POD reagent enzymes. Freeze-dried powder

of glucose oxidase (GOD), peroxidase (POD) and 4-

aminoantipyrine. Stable for 5 years at -20 °C.

Dissolve the contents of one bottle 4 in approx. 20 mL of

solution 3 and quantitatively transfer this to the bottle

containing the remainder of solution 3. Cover this bottle with

aluminum foil to protect the enclosed reagent from light.

Stable for 3 months at 2-5 °C or 12 months at -20 °C.

Solution 5. D-Glucose standard solution (5 mL, 1.0 mg/mL)

in 0.2% (w/v) benzoic acid. Stable for 5 years at room

temperature.

Safety

The general safety measures that apply to all chemical

substances should be followed. For more information

regarding the safe usage of this kit please refer to MSDS

available at www.nzytech.com.

D-glucose + O2 + H2O D-Gluconate + H2O2

2 H2O2 + p-hydroxybenzoic acid + 4-aminoantipirine

Quinoneimine dye + 4 H2O

Hydrolysis of sucrose (at pH 4.6)

Sucrose + H2O D-Glucose + D-Fructose -fructosidase

GOx

POD

Procedure (endpoint analysis)

Wavelength: 510 nm

Cuvette: 1 cm light path (glass or plastic)

Temperature: 50 °C

Final volume: 3.3 mL

Sample solution: 10-100 μg of total sucrose and D-glucose

per cuvette

Read against reagent blank

Figure 1. Linearity of the assay. Standard curves relating D-Glucose

and Sucrose concentration (g/test) to absorbance at 510 nm (25 ºC)

Mixtures can be obtained with a plastic spatula or by gentle inversion after sealing with a cuvette cap or Parafilm

®.

*Pipette both Solution 1+2 and sample into the bottom of the cuvette and mix by gentle swirling

Calculation

The concentration of D-glucose and sucrose (g/L) are

calculated as follows:

Sucrose calculation takes in account the conversion of µg of

D-Glucose (as measured) to µg of sucrose

If the sample has been diluted or a different sample volume

was used during the reaction, the result must be multiplied

by the corresponding dilution/concentration factor.

Interferences

If the concentration of D-glucose in the sample is much

larger than D-fructose (e.g. 10x higher), then the precision of

sucrose and D-fructose is compromised. In this situation, the

content of D-glucose should be reduced using glucose

oxidase/catalase reagent in the presence of oxygen (see

Sample Preparation)

An internal standard should be included during sample

analysis if the presence of interfering substances is

suspected. A quantitative recovery of this standard should be

expected.

With each new batch of GODPOD Reagent, the time of

maximum colour formation with 100 µg of D-Glucose

standard should be checked. This is approximately 15 min.

(See Figure 2)

0.00

0.20

0.40

0.60

0.80

1.00

0 20 40 60 80 100

Pipette into cuvettes (mL) Blank D-Glucose

Standard

For each sample

D-Glucose assay

(A)

Sucrose + D-Glucose

assay (B)

Solution 1+2*

Sample

Solution 1

H2O

-

-

-

0.30

-

0.10

-

0.20

-

0.10

0.20

-

0.20

0.10

-

-

Mix. Incubate for 20 min at 50 ºC. Then add:

GODPOD reagent (3+4) 3.00 3.00 3.00 3.00

Mix, incubate at 50 °C for 20 min and read absorbances at 510 nm against the reagent blank to obtain ΔAD-glucose standard,

ΔAsample A and ΔAsample B

[g/L]

[g/L]

Ab

sorb

ance

@ 5

10

nm

g/test

Sucrose

D-Glucose

Figure 2. Colour formation on incubation of 100 g of D-glucose,

performed with NZYTech kit at 25 ºC using 1 cm pathlength

cuvettes. Time for maximum colour formation: 15 min.

General information on sample preparation

The total amount of sucrose and D-glucose present in the

cuvette should range between 10 and 100 μg. Thus, if a

sample volume of 0.10 mL is used the sample solution must

be diluted to yield a sugar concentration between 100 and

1000 mg/L.

To implement this assay use clear, colourless and practically

neutral liquid samples directly, or after dilution; filter turbid

solutions; degas samples containing carbon dioxide (e.g. by

filtration; measure "coloured" samples (against a sample

blank; treat "strongly coloured" samples that are used

undiluted or with a higher sample volume with PVPP (ad 0.2

g of PVPP/10 mL sample, shake vigorously for 5 min and

filter through Whatman nº1 filter paper); crush or

homogenize solid or semi-solid samples, extract with water

or dissolve; extract samples containing fat with hot water.

Examples of sample preparation

Determination of sucrose and D-glucose in fruit juices

Filter turbid juices or clarify using Carrez reagents. Dilute to

give a sugar concentration of approximately 0.1-1.0 g/L.

Strong coloured samples should be treated with PVPP (see

above). Slightly coloured samples can be assayed directly.

Typically, for orange and apple juice, a dilution of 1:100 and a

sample volume of 0.1 mL are satisfactory.

Determination of sucrose and D-glucose in honey

After stirring the honey sample thoroughly with a spatula,

transfer approx. 10 g of the viscous or crystalline material to

a beaker and heat for 15 min at approx. 60 °C and stir (there

is no need to stir liquid honey). After cooling, prepare 1%

(w/v) honey solution: pour approx. 1 g of the liquid sample

accurately weighed, into a 100 mL volumetric flask. Dissolve

initially with only a small volume of distilled water and then

dilute to the mark and mix.

Determination of D-glucose: typically, a dilution of 1:10 of

the 1% (W/V) honey solution and a sample volume of 0.1

mL are satisfactory.

Determination of sucrose: if the estimated sucrose

concentration of the honey lies between 5 and 10%, a

dilution 1:3 of the 1% (W/V) honey solution and sample

volume of 0.1 mL are satisfactory. If the estimated sucrose

concentration of the honey lies below 5%, D-glucose

should be removed (see below), otherwise the precision of

the sucrose determination will be compromised.

Determination of sucrose and D-glucose in jam

Homogenise about 10 g of jam in a mixer. Pour approx. 0.5 g

of the sample accurately weighed, into a 100 mL volumetric

flask. Dissolve initially with only a small volume of distilled

water (50 mL) and then dilute to the mark, mix and filter.

Discard the first 5 mL of filtrate. Typically, no dilution will be

required and a sample volume of 0.1 is satisfactory.

Special sample preparation for the determination of

sucrose and D-fructose in the presence of excess of D-

glucose

Pipette to a 25 mL volumetric fask: 5 mL of buffer (300 mM

sodium phosphate plus 5 mM MgCl2, pH 7.6), 5 mL of sample

solution and 0.2 mL of enzyme solution (glucose oxidase 600

U/mL plus catalase 15000 U/mL). Incubate at 25 ºC and pass

a current air (O2) through the mixture for 1 h. After the

reaction, inactivate the enzymes by incubation the flask in a

boiling water bath for 10 min. After cooling, dilute the

content to the mark with distilled water. Mix and filter. Use

0.5 mL of the clear solution for the determination of sucrose.

Determine residual D-glucose as habitual.

References

Outlaw, W. H. & Mitchell, C. T. (1988). Sucrose. In Methods of

Enzymatic Analysis (Bergmeyer, H. U., ed.), 3rd ed., Vol. VI, pp.

96-103, VCH Publishers (UK) Ltd., Cambridge, UK.

Kunst, A., Draeger, B. & Ziegenhorn, J. (1988). D-Glucose. In:

Methods of Enzymatic Analysis (Bergmeyer, H. U., ed.), 3rd

ed., Vol.VI, pp. 163-172, VCH Publishers (UK) Ltd., Cambridge,

UK.

Released 01/16

0.00

0.20

0.40

0.60

0.80

1.00

0 5 10 15 20 25

Incubation time

(min)

Ab

sorb

ance

@ 5

10

nm

Maximum colour formation

Please enquire info@nzytech.com to obtain any additional information about this kit,

including additional specific applications.

Estrada do Paço do Lumiar, 22

Campus do Lumiar - Edifício E, R/C

1649-038 Lisboa, Portugal Tel.:+351.213643514

Fax: +351.217151168 www.nzytech.com

Certificate of Analysis

Test Criteria Result

Test Performance Reaction completed within time stated Meets specification

Target value for recommended standard material +/- 10% Meets specification

Blank reaction absorbance +/- 10% of the blank value Meets specification

Approved by:

José Prates

Senior Manager, Quality Systems